Method for determining plasma protein binding rate of polypeptide coupled radionuclide drug
By combining trichloroacetic acid precipitation method and γ-counter, the uncertainty and complexity of the determination of plasma protein binding rate of polypeptide-coupled radionuclide drugs in the prior art were solved, and the measurement effect with high accuracy and specificity was achieved.
Patent Information
- Application Number
- CN202411735299.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-03-25
AI Technical Summary
The existing methods for determining plasma protein binding rate of polypeptide-coupled radionuclide drugs have uncertainties, complexity and experimental errors, making it difficult to achieve high accuracy and specificity assays.
The trichloroacetic acid precipitation method is combined with the γ-counter. The protein is gradually separated and the unbound drugs are removed through two concentrations of trichloroacetic acid solution to ensure that the radioactive count in the precipitation mainly comes from the drugs bound to the protein.
The efficient and accurate determination of the plasma protein binding rate of the drug is achieved, which improves the accuracy and specificity of the determination and reduces experimental errors.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of drug analysis, and in particular relates to a method and system for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug. Background Art
[0002] Peptide-coupled radionuclide drugs are radionuclides that are used as payloads and chelated with chelators before being coupled to peptides through linkers to form peptide-coupled radionuclides. This guides the radionuclides to precise targeting, and combined with the advantages of peptides such as good tissue penetration and low immunogenicity, it can achieve ideal drug delivery in the body, thereby reducing toxicity and increasing efficacy.
[0003] In recent years, a number of peptide-coupled radionuclide drugs have been approved. They can be divided into diagnostic and therapeutic types according to the radionuclide particles they emit and their uses. They have played an increasingly important diagnostic and therapeutic role in the field of tumor diseases and have broad application prospects.
[0004] Plasma protein binding rate is an important parameter in drug research, which affects the biological activity, distribution, metabolism and clearance rate of drugs, and thus affects the efficacy and safety of drugs. In the process of drug development, especially in the development of radionuclide drugs, the accurate determination of plasma protein binding rate is crucial for the preclinical evaluation of drugs.
[0005] Common methods for determining the plasma protein binding rate of drugs include equilibrium dialysis, ultracentrifugation, ultrafiltration, etc., combined with analytical instruments such as high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS / MS) to detect the concentration of drugs in samples. The analytical method needs to be optimized and developed, and the analysis time is relatively long.
[0006] However, the existing determination methods have certain uncertainties in operation and may be interfered by other components in the plasma sample. Specifically, there may be a large number of endogenous interfering substances in the peptide molecule organism, and the analysis method needs to have high specificity and sensitivity; peptide molecules are prone to form multiple charges in the mass spectrometry ion source, and ions of different valence states are relatively dispersed, so it is difficult to find the optimal ion pair, which brings great challenges to sample processing and detection.
[0007] At the same time, the conventional method is relatively complicated, the operation process is cumbersome, and experimental errors are prone to occur. Therefore, a simple, accurate, and reproducible method for determining the plasma protein binding rate is urgently needed. Summary of the invention
[0008] The purpose of the present invention is to solve the deficiencies in the prior art and to provide a method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug by optimizing the experimental steps and improving the accuracy of the determination of the plasma protein binding rate; and a plasma protein binding rate determination system based on the method.
[0009] In order to solve the above technical problems, the technical method adopted by the present invention is as follows: the present invention discloses a method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug, comprising the following steps:
[0010] Step S1, thawing the frozen blank plasma in cold water, removing the upper and bottom materials by centrifugation, and measuring the pH value of the plasma; diluting the test solution to an appropriate concentration, adding the blank plasma to the test solution and mixing thoroughly to obtain a plasma sample of the required concentration, and incubating it in a constant temperature water bath at 37°C for a certain period of time;
[0011] Step S2, adding a trichloroacetic acid solution of a certain concentration to the test tube, centrifuging and collecting the supernatant; then adding a trichloroacetic acid solution of a certain concentration to the precipitate, centrifuging and collecting the supernatant, repeating this step several times, combining the collected supernatants and collecting the precipitate separately;
[0012] Step S3, using a γ-counter to measure the radioactive counts of the background, supernatant and precipitate, respectively, and calculating the plasma protein binding rate of the drug by the following calculation formula:
[0013]
[0014] Furthermore, in step S1, the blank plasma is centrifuged at 3500 rpm and 4°C for 5 minutes to remove the upper and lower layer substances. Furthermore, in step S1, the pH value of the blank plasma is between 7.0 and 8.0. If it is not within this range, the pH value is adjusted using 1% phosphoric acid solution or 1M sodium hydroxide solution.
[0015] Furthermore, the radioactivity of the sample stock solution was measured using a medical activity meter, and the sample solution was diluted to about 2 μCi / 100 μL according to the radioactivity.
[0016] Furthermore, in step S1, the volume ratio of the plasma sample to the test solution is 1:1, the mixture is evenly mixed and incubated in a constant temperature water bath at 37° C. for 2 hours.
[0017] Furthermore, in step S2, 1 mL of 250 g / L trichloroacetic acid was added to each test tube, and the mixture was centrifuged at 2000 rpm for 5 minutes to collect the supernatant;
[0018] Then, 1 mL of 100 g / L trichloroacetic acid was added to the precipitate, and the mixture was centrifuged at 2000 rpm for 5 minutes to collect the supernatant. This step was repeated twice, and the collected supernatants were combined and the precipitate was collected separately.
[0019] Furthermore, the preparation method of the trichloroacetic acid solution is: weigh 12.5g of ammonium acetate, add 50.0mL of pure water and mix well to prepare a trichloroacetic acid solution with a concentration of 250g / L; weigh 5.00g of ammonium acetate, add 50.0mL of pure water and mix well to prepare a trichloroacetic acid solution with a concentration of 100g / L.
[0020] Furthermore, in step S1, a molecular sieve or affinity chromatography material is further added to the plasma sample to improve the separation effect of the drug and plasma protein, thereby enhancing the determination specificity of the drug-protein binding.
[0021] Furthermore, in step S2, the background count is performed by measuring the blank test tube in parallel 6 times and taking the average value.
[0022] The present invention also discloses a system for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug, comprising:
[0023] A sample processing module, used to prepare and process the test sample and blank plasma, and prepare the sample according to step S1;
[0024] A protein separation and precipitation module, including a trichloroacetic acid solution and a centrifugal device, for performing protein precipitation treatment on the plasma sample according to step S2;
[0025] A radioactive counting module, used for measuring radioactive counts, comprising a γ-counter, for measuring the radioactive counts of the sample and calculating the plasma protein binding rate according to step S3;
[0026] The data calculation module is used to calculate the plasma protein binding rate of the drug according to the radioactive counting result and execute the calculation formula described in step S3.
[0027] Beneficial effects:
[0028] 1. The trichloroacetic acid precipitation method used in the present invention is combined with the radioactive counting method to efficiently and accurately determine the plasma protein binding rate of the drug. In the separation and precipitation process of the protein, the innovative points of the experimental method include: gradually separating the protein and removing the unbound drug by two concentrations of trichloroacetic acid solution. This two-step separation method ensures that the radioactive count in the precipitation mainly comes from the drug bound to the protein, while the radioactive drug in the supernatant is effectively removed. This technical innovation is of great significance for improving the accuracy and specificity of the plasma protein binding rate determination.
[0029] 2. The plasma protein binding rate is determined by the trichloroacetic acid precipitation method combined with a γ-counter. By using a γ-counter for radioactivity counting, the present invention can be applied to a variety of different types of drugs, and is particularly suitable for the determination of the plasma protein binding rate of polypeptide-coupled radionuclide drugs. Due to the special properties of polypeptide-coupled radionuclide drugs, this method can effectively determine their binding behavior in plasma.
[0030] 3. In this determination method, the pH value of plasma, the concentration of trichloroacetic acid solution, centrifugation conditions and other factors were finely adjusted to ensure the accuracy of the experimental results. This meticulous optimization of experimental conditions reflects the technical maturity of the method of the present invention and its adaptability to complex experimental conditions. DETAILED DESCRIPTION
[0031] The following example is used to illustrate in detail the method for determining the plasma protein binding rate of the polypeptide-coupled radionuclide drug of the present invention. This example shows the actual operation steps, including sample preparation, plasma protein separation, radioactivity counting and calculation of the plasma protein binding rate.
[0032] Example 1: A method for determining the plasma protein binding rate of drug A
[0033] The equipment used in this embodiment is as follows:
[0034]
[0035]
[0036] 1. Preparation of test drug solution:
[0037] 1.1 Preparation of test drug A solution:
[0038] First, a radioactivity meter (CAPINTEC INC; CRC-25R) was used to measure the radioactivity of the drug A stock solution, which was 18.7 mCi and the volume of the stock solution was 1 mL.
[0039] 10.7 μL of drug A stock solution was taken and added to a centrifuge tube containing 989.3 μL of sodium chloride injection to obtain stock 1 solution.
[0040] Pipette 500 μL of stock 1 solution and add it to a centrifuge tube containing 4500 μL of sodium chloride injection to finally obtain a suitable test solution with a solution concentration of approximately 2 μCi / 100 μL.
[0041] 1.2 Preparation of trichloroacetic acid solution:
[0042] Preparation of 250g / L trichloroacetic acid solution: Weigh 12.5g of ammonium acetate, add it into 50.0mL of pure water and mix well to obtain a 250g / L trichloroacetic acid solution. Store it at room temperature and it is valid for three days from the date of preparation.
[0043] Preparation of 100g / L trichloroacetic acid solution: Weigh 5.00g of ammonium acetate, add it into 50.0mL of pure water and mix well to obtain a 100g / L trichloroacetic acid solution. Store it at room temperature and it is valid for three days from the date of preparation.
[0044] 2. Preparation and Incubation of Plasma Samples
[0045] 2.1 Thawing and centrifugation of plasma:
[0046] Frozen rat blank plasma was thawed in cold water.
[0047] The supernatant and bottom layers were removed by centrifugation at 3500 rpm and 4°C for 5 minutes using a high-speed refrigerated centrifuge (Eppendorf Centrifuge 5424R). The plasma pH was measured and ensured to be between 7.0 and 8.0.
[0048] 2.2 Mixing of plasma and drug solution:
[0049] After diluting the test drug solution (as described in step 1) to an appropriate concentration, take an appropriate amount of blank plasma and add it to the test solution and mix thoroughly to obtain a plasma sample of the desired concentration.
[0050] The plasma sample was mixed with the test solution in a 1:1 volume ratio and incubated in a 37°C constant temperature water bath for 2 hours.
[0051] 3. Protein precipitation and separation
[0052] 3.1 Protein precipitation operation:
[0053] Take 3 test tubes, add 100 μL of plasma sample and 100 μL of test solution (about 2 μCi of drug) to each test tube, mix well, and incubate in a 37°C constant temperature water bath for 2 hours.
[0054] 1 mL of 250 g / L trichloroacetic acid solution was added to each test tube, and the tubes were centrifuged at 2000 rpm for 5 minutes using an Eppendorf Centrifuge 5424R high-speed centrifuge to collect the supernatant.
[0055] The precipitate was added with 1 mL of 100 g / L trichloroacetic acid solution, centrifuged again at 2000 rpm for 5 minutes, and the supernatant was collected and labeled as SD rat-supernatant-1. This step was repeated twice, labeled as SD rat-supernatant-2 and SD rat-supernatant-3, and finally the supernatants were combined and the precipitates were collected separately, labeled as "SD rat-precipitate-1", "SD rat-precipitate-2" and "SD rat-precipitate-3".
[0056] 3.2 Radioactivity Count:
[0057] The radioactivity counts of background, supernatant and precipitate were measured using a PerkinElmer γ-counter (2480WIZARD2). Three parallel measurements were performed for each sample to ensure the reliability of the results.
[0058] 4. Calculation of plasma protein binding rate
[0059] The plasma protein binding rate was calculated based on the measured radioactivity count data using the following formula:
[0060] PPB (%) = (precipitation count - background count) / [(precipitation count - background count) + supernatant count - background count)] × 100%
[0061] Test results
[0062]
[0063] In the above experiment, the radioactivity of plasma samples after different treatment steps was measured by a standard radioactivity counting method, and the plasma protein binding rate of drug A was calculated by a calculation formula.
[0064] The experimental results showed that the plasma protein binding rate of drug A was 54.15%, and the repeatability was good, with a standard deviation (SD) of 0.40 and a coefficient of variation (%CV) of 0.74%.
[0065] The experimental results showed that the plasma protein binding rate of drug A was 54.15% (n=3, mean of three parallel experiments). This value indicates that drug A has a moderate degree of protein binding in plasma.
[0066] The radioactivity value of the background count was 236.92 CPM, while the radioactivity counts of the supernatant and precipitate during the experiment were significantly higher than this value, reaching hundreds of thousands of CPM (e.g., 173957-176339 CPM for the supernatant and 204738-208531 CPM for the precipitate). This difference indicates that during the plasma sample processing, the binding of the drug to the plasma protein was effectively separated, and the trichloroacetic acid precipitation method successfully removed the unbound drug, ensuring the specificity and accuracy of the determination.
[0067] It can be seen from the data that the radioactivity count of the protein precipitate is significantly higher than that of the supernatant, which further confirms that the trichloroacetic acid precipitation method effectively recovers the drug components bound to plasma proteins. The step-by-step centrifugation and solution optimization (such as trichloroacetic acid at concentrations of 250 g / L and 100 g / L) used in the experimental design can ensure the effective separation of drugs and proteins, thereby improving the determination accuracy of plasma protein binding rate.
[0068] Based on the above, the following conclusions can be drawn:
[0069] 1. The trichloroacetic acid precipitation method used in the present invention is combined with the radioactive counting method to efficiently and accurately determine the plasma protein binding rate of the drug. In the separation and precipitation process of the protein, the innovative points of the experimental method include: gradually separating the protein and removing the unbound drug by two concentrations of trichloroacetic acid solution. This two-step separation method ensures that the radioactive count in the precipitation mainly comes from the drug bound to the protein, while the radioactive drug in the supernatant is effectively removed. This technical innovation is of great significance for improving the accuracy and specificity of the plasma protein binding rate determination.
[0070] 2. The plasma protein binding rate is determined by the trichloroacetic acid precipitation method combined with a γ-counter. By using a γ-counter for radioactivity counting, the present invention can be applied to a variety of different types of drugs, and is particularly suitable for the determination of the plasma protein binding rate of polypeptide-coupled radionuclide drugs. Due to the special properties of polypeptide-coupled radionuclide drugs, this method can effectively determine their binding behavior in plasma.
[0071] 3. During the experiment, the pH value of plasma, the concentration of trichloroacetic acid solution, centrifugation conditions and other factors were finely adjusted to ensure the accuracy of the experimental results. This meticulous optimization of experimental conditions reflects the technical maturity of the method of the present invention and its adaptability to complex experimental conditions.
[0072] Example 2 - System for determining plasma protein binding rate of peptide-coupled radionuclide drugs
[0073] This embodiment provides a system for determining the plasma protein binding rate of a peptide-coupled radionuclide drug. The system integrates sample preparation, protein separation, radioactivity counting and data analysis modules to achieve a fully automated and standardized plasma protein binding rate determination process.
[0074] 1. System Overview
[0075] The system includes the following modules:
[0076] Sample processing module: used for the preparation of test solution and blank plasma, and performs sample pretreatment and mixing according to step S1.
[0077] The protein separation and precipitation module includes a trichloroacetic acid solution and a centrifugal device, and is used to perform protein precipitation of the plasma sample according to step S2.
[0078] Radioactivity counting module: used to measure the distribution of radiopharmaceuticals in samples, including a γ-counter for counting the radioactivity in the supernatant and precipitate.
[0079] Data calculation module: used to calculate the plasma protein binding rate according to the radioactivity counting results and execute the calculation formula in step S3.
[0080] 2. System composition
[0081] The specific composition of the system is as follows:
[0082] 2.1 Sample processing module
[0083] Function: This module is responsible for providing sample preparation functions, including dilution of test solution and preparation of plasma samples.
[0084] Main equipment:
[0085] Pipette (Eppendorf Research plus): For precise aspiration and transfer of liquid samples.
[0086] Constant temperature water bath (Joanlab BHS-2): used to provide a constant temperature environment to ensure that the samples are incubated at 37°C.
[0087] Centrifuge (Eppendorf Centrifuge 5424R): used to process plasma samples and remove the upper and lower layers.
[0088] 2.2 Protein separation and precipitation module
[0089] Function: This module separates drugs from plasma proteins by using trichloroacetic acid precipitation to separate proteins and removing unbound drugs by centrifugation.
[0090] Main equipment:
[0091] High-speed refrigerated centrifuge (Eppendorf Centrifuge 5424R): This device is used for protein precipitation and separation of drug-protein bound parts during centrifugation.
[0092] Trichloroacetic acid solution preparation device: used to prepare 250g / L and 100g / L trichloroacetic acid solutions to ensure high protein precipitation efficiency.
[0093] 2.3 Radioactivity counting module
[0094] Function: This module is used to measure the distribution of radioactive drugs in samples, using a γ-counter to count the radioactivity of the supernatant and precipitate.
[0095] Main equipment:
[0096] Radioactivity Counter (PerkinElmer 2480WIZARD2): This device is used to accurately measure the content of radioactive drugs in samples. By measuring the radioactivity counts of the supernatant and the precipitate, the system can effectively calculate the plasma protein binding rate of the drug.
[0097] 2.4 Data Calculation Module
[0098] Function: Based on the data obtained from the radioactivity counting module, the plasma protein binding rate of the drug is calculated using a predetermined formula. The calculation results will be automatically output and displayed.
[0099] Main equipment:
[0100] Computer system and software: used to run the calculation formula and generate an analysis report of plasma protein binding rate. This module can automatically perform formula calculations and output detailed experimental data and statistical analysis.
[0101] 3. System workflow
[0102] Sample preparation:
[0103] Prepare the test solution through the sample processing module and mix the blank plasma and the test solution.
[0104] The samples were incubated in a 37°C constant temperature water bath for 2 h to ensure that the drug was fully bound to plasma proteins.
[0105] Protein separation:
[0106] The incubated samples were transferred to centrifuge tubes and trichloroacetic acid solution was added for protein precipitation.
[0107] After three centrifugations, the supernatants were collected and combined to separate the precipitate.
[0108] Radioactivity count:
[0109] The radioactivity of the supernatant and the precipitate was counted using a γ-counter, and the radioactivity distribution of the drug was calculated.
[0110] Data analysis and calculation:
[0111] The data calculation module calculates the plasma protein binding rate through a formula based on the radioactive counting results.
[0112] The system automatically outputs the calculation results and generates an experimental report.
[0113] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.
Claims
1. A method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug, characterized in that: The following steps are involved: Step S1, thawing the frozen blank plasma in cold water, removing the upper and bottom materials by centrifugation, and measuring the pH value of the plasma; diluting the test solution to an appropriate concentration, adding the blank plasma to the test solution and mixing thoroughly to obtain a plasma sample of the required concentration, and incubating it in a constant temperature water bath at 37°C for a certain period of time; Step S2, adding a trichloroacetic acid solution of a certain concentration to the test tube, centrifuging and collecting the supernatant; then adding a trichloroacetic acid solution of a certain concentration to the precipitate, centrifuging and collecting the supernatant, repeating this step several times, combining the collected supernatants and collecting the precipitate separately; Step S3, using a γ-counter to measure the radioactive counts of the background, supernatant and precipitate, respectively, and calculating the plasma protein binding rate of the drug by the following calculation formula:
2. The method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug according to claim 1, characterized in that: In step S1, the blank plasma is centrifuged at 3500 rpm and 4° C. for 5 minutes to remove the upper and lower layer materials.
3. The method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug according to claim 1, characterized in that: In step S1, the pH value of the blank plasma is between 7.0 and 8.
0. If it is not within this range, a 1% phosphoric acid solution or a 1M sodium hydroxide solution is used to adjust the pH value.
4. The method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug according to claim 1, characterized in that: The radioactivity of the sample stock solution was measured using a medical activity meter, and the sample solution was diluted to about 2 μCi / 100μL.
5. The method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug according to claim 1, characterized in that: In step S1, the volume ratio of the plasma sample to the test solution is 1:1, the mixture is mixed evenly and incubated in a constant temperature water bath at 37° C. for 2 hours.
6. The method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug according to claim 1, characterized in that: In step S2, 1 mL of 250 g / L trichloroacetic acid was added to each test tube, and the mixture was centrifuged at 2000 rpm for 5 minutes to collect the supernatant; Then, 1 mL of 100 g / L trichloroacetic acid was added to the precipitate, and the mixture was centrifuged at 2000 rpm for 5 minutes to collect the supernatant. This step was repeated twice, the collected supernatants were combined, and the precipitates were collected separately.
7. The method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug according to claim 1, characterized in that: The preparation method of the trichloroacetic acid solution is as follows: weigh 12.5 g of ammonium acetate, add 50.0 mL of pure water and mix well to prepare a trichloroacetic acid solution with a concentration of 250 g / L; weigh 5.00 g of ammonium acetate, add 50.0 mL of pure water and mix well to prepare a trichloroacetic acid solution with a concentration of 100 g / L.
8. The method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug according to claim 1, characterized in that: In the step S1, a molecular sieve or affinity chromatography material is further added to the plasma sample to improve the separation effect of the drug and plasma protein, thereby enhancing the determination specificity of the drug-protein binding.
9. The method for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug according to claim 1, characterized in that: In step S2, the background count is measured in parallel 6 times in a blank test tube and the average value is taken.
10. A system for determining the plasma protein binding rate of a polypeptide-coupled radionuclide drug, characterized in that: According to any one of claims 1 to 9, the system comprises: A sample processing module, used to prepare and process the test sample and blank plasma, and prepare the sample according to step S1; A protein separation and precipitation module, including a trichloroacetic acid solution and a centrifugal device, for performing protein precipitation treatment on the plasma sample according to step S2; A radioactive counting module, used for measuring radioactive counts, comprising a γ-counter, for measuring the radioactive counts of the sample and calculating the plasma protein binding rate according to step S3; The data calculation module is used to calculate the plasma protein binding rate of the drug according to the radioactive counting result and execute the calculation formula described in step S3.